The platelet histocompatibility type and anti-platelet immune status of an individual both play important roles in the management of clinical conditions due to anti-platelet immune response. Anti-platelet immune responses resulting in the production of platelet-destroying antibodies occur as a result of, among other things, pregnancy, platelet transfusion therapy and anti-platelet autoimmune disease.
Of particular interest to the present invention is platelet transfusion therapy. Since its beginning over three-quarters of a century ago, platelet transfusion therapy has played an increasingly important role in the supportive care of patients with bone marrow failure. However, one impediment to progress in this field has been the finding that a significant proportion of recipients become refractory to repeated transfusions from random donors. Platelet rejection appears to be due in many cases to anti-platelet antibodies in the transfusion recipient (donee) induced as a result of alloimmunization.
Alloimmunization is the process wherein an individual produces antibodies in response to exposure to an alloantigen. An alloantigen is an antigen, typically a protein, that exists in alternative (allelic) forms in different individuals of the same species, and thus induces an immune response when one form is transferred (as by transfusion or tissue graft) to members of the same species who have not previously been exposed to it. Class I human leukocyte antigens (HLA) are one group of alloantigens that are found on the surface of platelets and are capable of inducing alloantibodies (antibodies against alloantigens) that mediate platelet rejection in transfusion recipients. Yankee et al. first demonstrated that transfusions of HLA-matched platelets from single donors frequently results in good increments in platelet count in transfusion recipients. That is, crossmatching of donor platelet and recipient HLA type has been found to reduce platelet rejection in transfusion recipients.
Unfortunately, HLA matched platelet donors are available for only a minority of alloimmunized patients, and the response of recipients to partially matched donor platelets is less predictable. The recognition of cross-reacting groups and the differential expression of certain HLA antigens (e.g., B12) on platelets has facilitated educated guessing during donor selection. However, a means of selecting donor platelets without resorting to trial and error would be advantageous.
Characterization of an individual's anti-platelet immune status is, as previously mentioned, also important in managing autoimmune diseases. One such disease is chronic immune thrombocytopenic purpura (ITP), a syndrome of destructive thrombocytopenia due to an antibody against a platelet-associated antigen (McMillan, N. Engl. J. Med. 304:1135-1147 (1981), and Kelton et al., Semin. Thromb. Haemost., 8:83-104 (1982). Van Leeuwen et al., (Blood, 59:23-26 (1982)) first provided evidence that autoantibodies were present in some ITP patients. They noted that of 42 antibody eluates from ITP platelets, 32 would bind to normal but not to thrombasthenic platelets; the remaining eluates bound to both. Since thrombasthenic platelets are deficient in platelet glycoproteins (GP) IIb and IIIa, they suggested that these ITP patients had autoantibodies to one of these glycoproteins.
Direct evidence for anti-glycoprotein autoantibodies in chronic ITP has been provided by subsequent studies using a variety of methods. Woods et al. showed binding of autoantibodies from ITP patients to the GPIIb/IIIa complex or to GPIb attached to microtiter wells with monoclonal antibodies and confirmed these observations by immunoprecipitation. See Woods et al., Blood, 63:368-375 (1984) and Woods et al., Blood, 64:156-160 (1984). Using the former method, they noted anti-GPIIb/IIIa or anti-GPIb autoantibodies in about 10% of patients, much less than the percentage observed by the indirect studies of van Leeuven et al. supra.
Other investigators also detected antiplatelet autoantibodies in chronic ITP patients using immunoblotting (Mason et al., Br. J. Haematol., 56:529-534 (1984) and Beardsley et al., J. Clin. Invest. 74:1701-1707 (1984)), immunoprecipitation, (Woods et al., Blood, 63:368-375 (1984), Woods et al., Blood, 64:156-160 (1984) and Devine et al., Blood, 64:1240-1245 (1984)), inhibition of murine monoclonal anti-GPIIb/IIIa antibody binding to ITP platelets (Varon et al., Proc. Natl. Acad. Sci. USA, 80:6992-6995 (1983)) and crossed immunoelectrophoresis (Szatkowski et al., Blood, 67:310-315 (1986)). Nugent et al. ("Proceedings of the INSERM Symposium on utilization of monoclonal antibodies for the understanding and detection of platelet activity." Amsterdam, Elsevier Science Publishers, 1986) and Asano et al., (Blood, 66:1254-1260 (1985)) have established human hybridomas from ITP lymphocytes which synthesize monoclonal antiplatelet antibodies. Some of these are specific for platelet glycoproteins (Nugent et al., supra).
Of the assays used for demonstrating antiglycoprotein autoantibodies in chronic ITP, the microtiter well assay (Woods et al., Blood, 63:368-375 (1984) and Woods et al., Blood, 64:156-160 (1984)) is most easily adaptable to clinical use. However, the low percentage of positive tests (about 10%) when compared to that of van Leeuwen et al. (about 76%) suggested that solubilization of the platelets prior to antibody sensitization may alter some of the epitopes. For this reason, an assay (immunobead assay) for antiglycoprotein autoantibodies was designed where platelets are sensitized prior to their solubilization to take advantage of the possibility that the epitopes expressed by the platelet surface antigens may remain more stable when bound to antibody. This assay can measure both platelet-associated and plasma autoantibodies.